Medulloblastoma is an undifferentiated, neuroepithelial tumor of the cerebellum and the most common malignant brain tumor in children. While the current medical practice has significantly improved survival rates for children with medulloblastoma, approximately one-third of these patients die, and 20% of survivors sustain severe neurological damage. The standard of care for medulloblastoma includes surgical resection followed by radiation therapy and chemotherapy. Although larger resections that effectively remove the tumor correlate with improved patient outcome, the surgeon must attempt to minimize damage to surrounding healthy brain tissue. To improve tumor visualization and localization, we aim to adapt a dual axis confocal (DAC) microscope to detect tumor margins and guide surgical resection, and to identify new reagents that can mark the margins with novel molecular probes for clear differention of tumor from normal tissue. The DAC microscope will be modified with a GRIN relay lens to reach into the cavity ofthe resected tumor and interrogate the margins for residual cancer. The imaging reagents that are being developed will include peptides and antibodies that we will refine for use as fluorescent probes that label mouse and human medulloblastoma. These reagents are selected based on data obtained by high throughput analyses including DNA microarays and phage display, and take advantage of commercially available reagents. Binding of these markers to tumor margins will be visualized using the refined confocal microscope that is based on a dual axis design and will be modified for optimal use for surgical resection. We will develop software to identify tumor margins using the microanatomic features of cancer and normal brain after staining with vital dyes. The modifications to the microscope include those that stabilize the system and enable multispectral analyses through a disposable tip. To test the microscope in the clinic we will first use a FDA-approved dye as a contrast agent. As we develop new reagents, we will also validate their utility in image-guided resections. We will also develop image-processing algorithms to aid in the discrimination of tumors from surrounding normal brain. The research proposed here will serve as a foundation for the use of molecular probes and advanced microscopy for precision image-guided resection of solid brain tumors in humans. Successful completion of the aims to this project will have a significant benfit for children suffering from medulloblastoma by prolonging disease-free survival and reducing the neurological deficits associated with tumor resection.